Vitis 45 (1), 29–36 (2006) Assessment of powdery mildew resistance of grape and Erysiphe necator pathogenicity using a laboratory assay J. P. PÉROS1), T. H. NGUYEN2), C. TROULET2), C. MICHEL-ROMITI2) and J. L. NOTTEGHEM2) 1) UMR DGPC and 2) UMR BGPI, INRA, Montpellier, France Summary sources of resistance and to assist breeding programs for cultivars being resistant to powdery mildew. Determination To develop a quantitative evaluation of grapevine resist- of host resistance was based on field observations (BOUBALS ance to powdery mildew and of pathogenicity of the causal 1961, DOSTER and SCHNATHORST 1985 a, STAUDT 1997), tests agent (Erysiphe necator), a spot inoculation method was on leaf disks (STEIN et al. 1985, WANG et al. 1995) or on in developed using detached leaves of potted plants. The per- vitro material (KITAO and DOAZAN 1990). The results obtained centage of inoculating spots leading to a colony and the by different methods were found to be correlated (LI 1993). mean diameter of colonies were determined to assess the However, data were mostly presented on discrete scales of host-pathogen relationships. Significant differences were disease rating. Continuous variables would be more appro- found between host cultivars and their ranking was associ- priate for quantitative analysis of the relationships between ated with that observed in the vineyard. There was a signifi- grapevine and E. necator. cant interaction between cultivar and replicate during the Moreover, quantitative assessment of the pathogenic- whole experiment indicating that the physiological state of ity of E. necator would enable to choose the most appropri- detached leaves is important. Aging of tissues was accom- ate isolates to rate host genotypes. The diversity of the panied by a gain in resistance that was considerably more pathogen with respect to its pathogenicity has been poorly marked in resistant cultivars. Only partly expanded leaves investigated. KITAO and DOAZAN (1990) reported differences of resistant cultivars that stopped expansion on the agar in colony diameter, length of latent period and production of medium supported the development of the fungus. Signifi- conidia between a few isolates from different European re- cant differences between E. necator isolates were also dem- gions. Some evidence for pathogenic specialization was onstrated, but these variations were less marked than those observed when pathogenicity was examined for isolates due to host cultivar and leaf position. Preliminary results obtained from a range of Parthenocissus and Vitis species obtained with different isolates from the two European ge- (GADOURY and PEARSON 1991). A quantitative method in a netic groups (A and B) indicate that, on average, group A is closed-system laboratory environment would thus be par- less pathogenic. ticularly useful to address several questions regarding the diversity of the pathogen. For instance, the marked genetic K e y w o r d s : powdery mildew, Vitis vinifera, Vitis hybrids, differences between the morphologically similar groups that rating method, ontogenic resistance, Erysiphe necator, Uncinula have been identified in European, Indian and Australian necator, pathogenicity. populations of E. necator (DÉLYE et al. 1997, STUMMER et al. 2000) suggest that these groups may differ in pathogenicity, Introduction but this possibility has not been explored. The objectives of this study were 1) to develop a method Powdery mildew caused by Erysiphe necator Schwein. to obtain a simple, quantitative assessment of the interac- (formally Uncinula necator (Schwein.) Burrill (BRAUN and tion between grapevine and E. necator and 2) to evaluate TAKAMATSU 2000) is an economically important disease of the potential of this method using a set of grapevine grapevine (BULIT and LAFON 1978, PEARSON 1988). Several cultivars with different levels of susceptibility and resist- asexual cycles of reproduction of the fungus occur during ance in the vineyard as well as a set of isolates differing in the growing season and may cause losses of yield and qual- host origin and genetic group. ity (GADOURY et al. 2001, CALONNEC et al. 2004, STUMMER et al. 2005) if there is no or incomplete chemical control. The most efficient way to minimize powdery mildew is Material and Methods to grow resistant cultivars, however, the elite cultivars origi- nate from Vitis vinifera which is the most susceptible host P l a n t m a t e r i a l : Four Vitis vinifera cultivars species. Monogenic resistance to E. necator has been stud- (Cabernet-Sauvignon, Carignan, Cinsault, Grenache) and ied in Muscadinia and its hybrids with Vitis vinifera (BOU- three resistant interspecific cvs (Villard Blanc, Villard Noir, QUET 1986, PAUQUET et al. 2001). In the Vitis genus resist- Jacquez) were used. Villard Blanc and Villard Noir have a ance appears to be due to several genes (BOUBALS 1961, complex genealogy involving several Vitis species with a FISHER et al. 2004). Methods are needed to identify valuable majority of V. rupestris and V. vinifera parentage (ANTCLIFF Correspondance to: Dr. J. P. PÉROS, UMR DGPC, INRA Equipe Génétique Vigne, 2 place Viala, 34060 Montpellier cedex, France. Fax: +33-4-9961-2064. E-mail: [email protected] 30 J. P. PÉROS et al. 1992). Jacquez probably results from a cross between leaves every 4-5 weeks under the conditions described V. aestivalis and V. vinifera (GALET 1988). For this study, above. plants were obtained from one-bud cuttings prepared in P a t h o g e n i c i t y a s s a y : Inoculum was produced spring from one-year old branches collected in the preced- on detached leaves of cv. Cabernet-Sauvignon inoculated ing winter and stored at 4 °C. Cuttings were placed at high with infected tissues and incubated for 2-3 weeks under the density in flat boxes filled with vermiculite and maintained conditions described above. Detached leaves of potted for 6-8 weeks in a growth chamber at 25 °C in the light plants were inoculated using a spot method. About 20-60 (12 h·d-1, 40 µmol quanta m-2 s-1) and 22 °C in the dark. Rooted conidia were taken from a leaf infected with powdery mildew cuttings were then transferred in 2 liter pots containing a using a glass needle. Inoculum was deposited on a healthy mixture of soil and vermiculite (2:1), fertilized (Osmocote, leaf by gentle contact with the leaf epidermis. On each in- Scotts Europe B.V., Heerlen, The Netherlands) and grown in oculated leaf, three separate spots were applied approxi- the greenhouse. Plants were regularly trimmed to allow the mately in the middle between the petiole insertion point and development of new shoots with suitable leaves. the lobe extremity, avoiding veins. Inoculated leaves were Leaves of potted plants were removed, washed in tap incubated under the conditions described above. Inocula- water and disinfected for 1.5 min in a 0.3 % solution of active tion was considered successful if the spot resulted in a colony chloride. After three rinses in sterile distilled water, leaves that sporulated. The diameter of the area that sporulated were dried between several layers of sterile paper. Petiole was measured under the binocular microscope 11 d (Experi- length was then reduced to 1.5-2 cm. Leaves with their up- ment 3) or 20 d (other experiments) after inoculation. per surface visible were placed on 8 g·l-1 agar medium in 9 cm E x p e r i m e n t a l d e s i g n s a n d a n a l y s e s : Five Petri dishes; the remaining part of the petiole was inserted different experiments were performed using complete block into the medium. designs (Tab. 2). Each experiment was replicated three times F u n g a l i s o l a t e s : The isolates of E. necator used and, depending on the experiment, the replicate was studied in this study originated from infected material collected in as a second or a third classifying factor. Isolate FA01 was southern France (Ardeche district) in 2000 and 2001. In this used in experiments 1 and 2. Cv. Cabernet-Sauvignon was region, a population study identified the two genetic groups used in experiment 4. Leaves were taken at 5 different posi- that exist in Europe (PÉROS et al. 2005), and the set of isolates tions: the first expanding leaf (position 1), the second ex- included 5 group A isolates and 4 group B isolates (Tab. 1). panding leaf (position 2), and the three following leaves Within the two d after collection in the field, infected tissues (positions 3-5). The different positions were compared in were brushed onto detached leaves of cv. Cabernet- experiment 2 and position 2 and 4 were compared in experi- Sauvignon. The inoculated leaves were then incubated for ment 4. The leaves used in experiment 1 and 5 were taken at 10-15 d at 25 °C under 16·h d-1 illumination (40 µmol quanta position 2 and those used in experiment 3 were taken at m-2 s-1). Either a single or a few conidia from the same co- position 1. Statistical analyses were performed using SAS nidial chain were then picked up using a glass needle under (version 8.1; SAS Institute, Cary, NC, USA). The numbers of the binocular microscope. Conidia were spotted on detached colonies that sporulated were compared with χ2 testing us- leaves from the greenhouse or on leaves of in vitro plants, ing the FREQ procedure. Analyses of variance for the diam- cv. Cinsault, cultured as described by PÉROS et al. (1998). eter of the colony were performed using the GLM procedure The process was repeated once. The isolates were main- in SAS and means were compared using paired t-tests or the tained on leaves of in vitro plants and subcultured on new CONTRAST statement.